One overall objective of this proposal is the characterization of the chemical and replicative properties of mitochondrial DNA. Mitochondrial DNA will be isolated and purified from Paramecia during different stages of the growth cycle. The purified DNA will be analyzed from many viewpoints. 1) With regard to the mode of replication, the DNA will be examined in CsC1-ethidium bromide gradients and in sucrose gradients as well as in the electron microscope to determine whether replication is via rolling circle, D-loop synthesis, Cairns form, etc., mechanisms. 2) Chloramphenicol and ethidium bromide will be used to enrich for terminal and replicative forms and these will be analyzed by partial renaturation, circularization and heteroduplexing, etc., to further elucidate the mode of DNA replication. 3) From microinjection studies, it is known that isolated mitochondria transferred to recipient paramecia cells can proliferate and function only within certain stocks or syngens. This "restriction" will be explored by determining the degree of homology between mitochondrial DNA isolated from these stocks using renaturation and alkaline CsCl gradient methods. The degree of methylation of these DNA's will also be measured to decide whether the restriction encountered here is similar to that found in certain bacteria and bacteriophage where methyl groups protect the DNA from endonucleolytic cleavage. Another objective of this proposal is the study of mitochondrial function and biogenesis. Here, attempts will be made to transfer purified Mt DNA from oe Paramecium to another. Should resistance to antibiotics be transferable to sensitive cells by purified Mt DNA, it may be possible to study the transfer of resistance using bacterial or yeast Mt gene resistant to antibiotics. And finally we will develop in vitro assays as a measure of mitochondrial function at different stages of the age cycle. The opportunity to attempt such studies on interprotist transformation and gene relationships in one of several advantages unique to Paramecia. It is hoped that many aspects of mitochondrial function will lead to a more basic understanding of organisms with respect to normal and abnormal development.